Analysis of sodium hyaluronate by size exclusion chromatography

Abstract

Hyaluronic acid, HA, is a naturally occurring linear polysaccharide comprised of N-acetyl glucosamine and glucuronic acid. It is ubiquitous in tissues where it serves numerous biological functions. HA is polydisperse having average molecular weights that range on the order of 105 to 10 7 daltons depending upon the tissue from which it is derived and its method of purification. As with any polymer preparation, the molecular weight distribution, MWD, is important to a complete understanding of the characteristics and performance of the preparation. Size exclusion chromatography, SEC, is the most commonly employed means to determine the MWD of a polymer preparation. It is shown that SEC cannot be relied upon for the accurate and reproducible analysis of the MWD of large MW HA. ^ Commercially available polymer standards used to calibrate SEC systems have hydrodynamic volumes that are considerably smaller than HA. HA analyzed under extreme conditions of NaCl concentration and pH that were shown to cause contraction of the molecule still had a hydrodynamic volume much greater than polymer standards. Conventional calibration with standards can be subject to error from extrapolation and unable to detect elution in the totally excluded volume. ^ Viscosity measurements in a dual capillary viscometer demonstrated shear thinning of large MW HA at shear rates that are common inside the plumbing of SEC systems and on line viscometers. A model of shear inside an SEC column is derived in terms of the flowrate, column diameter, and particle diameter. The shear inside the column is shown to be sufficient to reduce the viscosity of large MW HA complicating the MWD analysis of HA by SEC. Chromatographic analysis based on separation in pressure driven flowing streams have fundamental limitations with shear sensitive polymers, such as HA, due to shear, which arises from parabolic flow profiles. Capillary electrophoretic methods have a flow profile that is flat and theoretically should have no shear. It is proposed that capillary electrochromatography utilizing a size exclusion stationary phase may provide the optimum tool for the analysis of large MW HA and other shear sensitive polymers. ^